This invention relates to assay techniques and to means for putting such techniques into effect. In particular it relates to an improved assay technique providing enhanced sensitivity and specificity.
The properties of simple optical structures have been known for many years and structures such as, for example, diffraction gratings have widespread use, not only as tools for understanding and analysing the wave properties of electromagnetic radiation but also, more recently, as sensors for detecting chemical, biochemical or biological species in a sample.
EP-0112721 describes the preparation, and use as biosensors, of optical structures having a pre-formed relief profile coated with a material (hereinafter called "specific binding partner") capable of binding with the species to be assayed (hereinafter called "ligand") to form a complex. The optical properties of such structures are altered as a result of complex formation between the ligand and the specific binding partner and this phenomenon can consequently form the basis of an assay system.
The change in optical properties of the structures described above is a function of the mass or bulk of the bound ligands, their dielectric properties and their optical characteristics such as refractive index and transparency. Hence, although the basic concept of utilising perturbed optical properties such as surface plasmon resonance (SPR) effects to form a sensor is valid, the practical applications of such methods have hitherto been limited because of poor sensitivity to small, low molecular weight ligands. This poor sensitivity precludes accurate measurement, at clinical concentrations, of low molecular weight ligands, for example with a molecular weight of less than 10,000 daltons.
A further difficulty experienced with the practical application of the technique described in EP-0112721 is that of poor specificity. This arises at two distinct stages of the assay procedure:
i) Non-specific binding of sample components to the surface of the optical structure. This can give rise to false positive results and inaccurate quantification of the perturbation of optical properties which arise as a result of specific ligand binding. PA1 ii) The use of only a single recognition step, i.e. the use of only one specific binding partner for the ligand to be analysed, can reduce specificity. Thus, where a ligand is present in a sample both as intact units and as fragments, e.g. parathyroid hormone, the use of only a single recognition step may result in both forms being bound when it may be desirable to measure only the intact, and hence biochemically active, molecules.
We have now devised an improved assay technique which can overcome the sensitivity disadvantages of the prior art and in some embodiments may also result in improved specificity.